Abstract: FR-PO697
High-Throughput, Prospective Discovery of Splice-Disruptive Variants (SDVs) in the Nephrotic Syndrome (NS) Gene WT1
Session Information
- Glomerular Diseases: Podocyte Biology - I
November 04, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
Abstract Time: 10:00 AM - 12:00 PM
Category: Glomerular Diseases
- 1304 Glomerular Diseases: Podocyte Biology
Authors
- Lai Yee, Jennifer, University of Michigan, Ann Arbor, Michigan, United States
- Smith, Cathy, University of Michigan, Ann Arbor, Michigan, United States
- Dunn, Ian Alexander, University of Michigan, Ann Arbor, Michigan, United States
- Burugula, Bala Bharathi B., University of Michigan, Ann Arbor, Michigan, United States
- Kitzman, Jacob O., University of Michigan, Ann Arbor, Michigan, United States
Background
SDVs contribute to a fraction of nearly every human genetic disorder. Frasier Syndrome, a syndromnic NS, is caused by SDVs near WT1 exon 9 splice donor site (SD), which decrease the ratio between two natural splice forms of WT1 called +KTS and -KTS. Beyond a few known Frasier Syndrome SDVs, accurately predicting other WT1 variants’ effects on splicing remains a challenge. In vitro minigene assays provide one means to test variant effect on splicing in highly multiplexed fashion. Thus, we coupled minigene assays with saturation mutagenesis across WT1 exon 9 to systematically identify WT1 SDVs in a high-throughput manner.
Methods
WT1 exon 9 plus 200 bases of the flanking introns were cloned into an established minigene plasmid, in between constant synthetic exons. Saturation mutagenesis was performed to generate a variant library including every single nucleotide variant across the cloned region, each associated with a unique barcode present in the constant downstream exon. This variant library was then transfected into 293T cells, and 24 hours later, RNA was harvested from those cells and spliced transcripts from the minigene library were read by target RNA-seq. The splicing patterns associated with each WT1 exon 9 variants were quantified from the aligned reads.
Results
Every possible single-base mutation was generated in the library, with a high degree of redundancy. Among spliced reads, +KTS and -KTS were the most highly represented isoforms and normally present at roughly 1:1 ratio. We identified 19 single-nucleotide variants near the +KTS SD which disrupt the normal splicing pattern of +KTS and -KTS isoform, favoring the expression of -KTS. Among these we detected all 8 known Frasier Syndrome variants. We also identify 16 single-nucleotide variants clustering near the -KTS SD which increased the expression of +KTS isoform.
Conclusion
Our saturation screen identifies all known Frasier syndrome SDVs in WT1 exon 9. We also nominate an additional 11 variants which similarly decrease the +KTS/-KTS ratio, and these represent possible yet unseen Fraiser Syndrome variants. We also discovered a set of variants which increase +KTS expression, which require further study. In summary, high throughput functional analyses can prospectively score genetic variants in NS and guide the functional classification
Funding
- Other NIH Support